ABSTRACT The Characterization Unit seeks a comprehensive understanding of molecular, cellular, and tissue changes that drive lung cancer development and progression during its very earliest stages. Lung cancer tumorigenesis occurs long before the development of clinically apparent disease, which affords the opportunity for intervention in this extended preclinical phase. Our multidisciplinary team will utilize state-of-the-art multi-omic technologies to address the key determinants that cause a pulmonary premalignant lesion (PML) to progress to invasive malignancy. In this first aim, we will utilize established assays to evaluate molecular and cellular alterations that contribute to lung carcinogenesis and pilot technologies in retrospective cohorts of lung premalignancy. Here we will address the evolution of premalignancy in the context of established cancers in cross-sectional analysis of both lung squamous (LUSC) and adenocarcinoma (LUAD) that have been previously banked from surgical specimens. The paramount challenge of delineating the determinants of progression or regression will be addressed in Aim 2 utilizing the molecular and cellular phenotyping of lung PMLs by employing established assays, and incorporating emerging higher resolution (single cell) technologies and successful pilot assays from Aim 1 in prospective cohorts. Here longitudinal prospective studies will allow temporal and spatial analysis of premalignancy. Precise clinical annotation will facilitate determination of critical translational outcomes. Because the premalignant microenvironment (PME) may either promote or constrain the development of invasive lung cancer, the Characterization Unit will evaluate the PME through extensive immune profiling. This investigation will facilitate subsequent analysis of the interplay between mutational load and gene expression status of premalignant cells and immune recognition, including identification of neoantigens and immunoediting, throughout the evolution of premalignancy. Using biospecimens procured by the Biospecimen Unit, the Characterization Unit will utilize technologies that yield multidimensional, large-scale data that can be analyzed by the Data Analysis Unit to discover relationships associated with disease progression. Technology platforms to be utilized to assess lung premalignancy and the corresponding microenvironment include: whole tissue (bulk) and single cell DNA- and RNA-sequencing, single nuclei RNA- sequencing, RNA in situ (SNAIL), highly multiplexed (40?50 markers/panel) imaging technologies including multiplexed ion beam imaging (MIBI), and CO-Detection by indEXing (CODEX) (including with Ce3D technology) in addition to validation by multiplex immunofluorescence (MIF). This Unit will ultimately provide the data that informs our understanding of the pathogenesis of lung cancer from its earliest appearance as premalignancy to invasive disease. This research will afford the opportunity to shift the paradigm from advanced disease intervention to early interception that prevents the progression of pulmonary premalignancy.